The interface between engineering and molecular life sciences has been fertile ground for advancing our understanding of complex biological systems. Engineered microstructures offer a diverse toolbox for cellular and molecular biologists to direct the placement of cells and small organisms, and to recreate biological functions in vitro: cells can be positioned and connected in a designed fashion, and connectivity and community effects of cells studied. Because of the highly polar morphology and finely compartmentalized functions of neurons, microfabricated cell culture systems and related on-chip technologies have become an important enabling platform for studying development, function and degeneration of the nervous system at the molecular and cellular level. Here we review some of the compartmentalization techniques developed so far to highlight how high-precision control of neuronal connectivity allows new approaches for studying axonal and synaptic biology.